Vaccination prevents severe COVID-19 outcome in patients with neutralizing type 1 interferon autoantibodies

Summary A hallmark of patients with autoimmune polyendocrine syndrome type 1 (APS-1) is serological neutralizing autoantibodies against type 1 interferons (IFN-I). The presence of these antibodies has been associated with severe course of COVID-19. The aims of this study were to investigate SARS-CoV-2 vaccine tolerability and immune responses in a large cohort of patients with APS-1 (N = 33) and how these vaccinated patients coped with subsequent infections. We report that adult patients with APS-1 were able to mount adequate SARS-CoV-2 spike-specific antibody responses after vaccination and observed no signs of decreased tolerability. Compared with age- and gender-matched healthy controls, patients with APS-1 had considerably lower peak antibody responses resembling elderly persons, but antibody decline was more rapid in the elderly. We demonstrate that vaccination protected patients with APS-1 from severe illness when infected with SARS-CoV-2 virus, overriding the systemic danger of IFN-I autoantibodies observed in previous studies.


INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID- 19) pandemic has been one the largest health threats in modern time. Over 662 million people have been affected so far, and official data report more than 6.7 million deaths worldwide. 1 As with influenza, several risk factors for severe disease have been identified, including age, obesity, and pre-existing disorders (reviewed in the study by Leretter et al. 2 ). Among the reported risk comorbidities are autoimmune disorders and immune deficiencies, in particular rare inborn errors of immunity (IEI). 2-4 One explanation for the increased risk is insufficient type 1 and type 2 interferon (IFN-I and IFN-II) production, which is essential for the antiviral response, 5-10 including monogenic disorders of genes involved in IFN-synthesis or the presence of neutralizing IFN-autoantibodies. Examples of the latter are myasthenia gravis, 11 autoimmune polyendocrine syndrome type I (APS-1), 12 immunodysregulation, polyendocrinopathy, enteropathy, X-linked, 13 and RAG mutation 14 syndromes. Surprisingly, it was also recently discovered that antibodies against IFN-I were quite common (4%) among persons over 70 years of age. 15 The presence of these antibodies correlates with severe COVID-19 illness, especially lung disease and fatal outcome. 15 Patients with APS-1, who have germline mutations in the autoimmune regulator (AIRE) gene, are of specific interest as almost all of these individuals have preexisting, extremely high levels of neutralizing antibodies against IFN-I. Lack of AIRE function leads to both autoimmune and immune deficiency components. [16][17][18] Indeed, several case studies have reported severe outcome and increased mortality for patients with APS-1 infected in the early waves of the COVID-19 pandemic before vaccination became widespread. 5, 6,[19][20][21] We hypothesized that IFN-I antibodies interfere with the adaptive immune response against SARS-CoV-2, although this was recently challenged by Sokal and coworkers who found that patients with APS-1 (at least young individuals) have adequate anti-SARS-CoV-2 humoral vaccine responses. 22 Hence, knowledge of the potential association of IFN-I antibodies and COVID-19 severity is still lacking. Previous studies were mostly conducted on small groups of unvaccinated patients early in the pandemic, infected with the Wuhan, Alpha  Taking advantage of one of the world's largest national registries and biobanks on patients with APS-1 (the Norwegian registry and biobank for organ-specific autoimmune disorders), we have here followed these patients longitudinally pre-and post-vaccination to investigate the tolerability and humoral specific immune responses to SARS-CoV-2 vaccination. We have also addressed in a cross-sectional design how SARS-CoV-2 infections affect vaccinated patients with APS-1.

COVID-19 vaccination is well tolerated in patients with APS-1
Patients Sixteen patients reported mild vaccine-associated adverse events, including headache, low-grade fever, and tiredness ( Figure 1D). We did not observe any specific pattern regarding adverse events related to type or timing of vaccines nor the number of vaccination doses received. One female patient who had three BNT162b2 doses experienced prolonged menstrual disorder with vaginal bleedings.
Patients with APS-1 have functional, although lower, SARS-CoV-2 antibody vaccine responses compared to healthy adults Serum SARS-CoV-2-specific (homologous Wuhan vaccine strain) antibody titers were measured by ELISA, Luminex, and virus neutralization assays pre, post, and in between three vaccine doses ( Figure 1). All patients, except two, had detectable ELISA and neutralization antibodies, although one additional patient did not have neutralizing antibodies ( Figures 1E, 1F, and S1). These results indicate that, in general, patients with APS-1 respond to vaccination by generating spike-specific antibodies. One patient on rituximab treatment still mounted an adequate vaccine response ( Figure S1). Individual longitudinal IgG-measurements of spike-1/spike-2/RBD/nucleocapsid also demonstrated an adequate vaccine response, and that the third booster dose maintained high anti-spike-IgG levels ( Figure S2). We additionally assayed 10 patients with APS-1 and two healthy controls at multiple time points (2-4) pre-and post-vaccination with monovalent vaccines against the original Wuhan strain (Table S2) for Omicron cross-reactive IgG antibodies. There were only three of the samples that had cross-reactive Omicron-neutralizing antibodies, two patients with APS-1 (one positive 47 days after third vaccination and one eight days after first vaccine) and one healthy control 112 days after third vaccination. In addition (* in Table S2), three patients were monitored after the initial study period and after confirmed infection by the SARS-2-CoV Omicron variant; two of these had higher Omicron-specific neutralizing antibodies while one remained negative.
We further measured autoantibodies against IFN-a2 and -u before and after SARS-CoV-2 vaccination in 10 patients with APS-1, to investigate whether the interferon autoantibodies were affected by vaccination. No substantial pre-to-post variations were observed, supporting the fact that these APS-1 hallmark autoantibodies might not interfere with humoral SARS-CoV-2 vaccine responses ( Figure S3).
We next compared the vaccine responses of patients with APS-1 with age-and gender-matched healthy controls (control cohort I) and an elderly cohort (control cohort II) after their first and second vaccine doses. As seen in Figure 2A, the peak responses of APS-1 patients were lower than age-matched controls, and The predicted curves for all subjects show that the models fit the data ( Figure S4). The small differences between the fitted and actual curves indicate that there is very little variation in the decay rates between subjects. However, we observed large differences in the initial vaccine response (at time 0, i.e. 14 days after first vaccine dose) between subjects. The panels were sorted by maximum estimated vaccine response at time 0; patients with APS-1 are mostly found at the bottom of the graph, indicating lower initial vaccine responses.

SARS-CoV-2 infections in vaccinated patients with APS-1
Twenty-four patients with APS-1 (66%) were infected by SARS-CoV-2 virus during the study period based on symptoms and a positive PCR or rapid antigen test (lateral flow test); all of these except one child were iScience Article infected post-vaccination ( Figures 3A and 3B). We confirmed that 19 of the patients with APS-1 had not been infected prior to vaccination with any measurable anti-nucleocapsid antibodies ( Figure S2). Infections occurred in the period December 2021-November 2022, and two patients had two confirmed SARS-CoV-2  [25]. Only symptoms that were reported in both studies and only for patients with APS-1 (*) are shown. Note that we did not ask our patients regarding taste/smell disturbances, appetite, headache, and sneezing and the reference paper did not describe concentration issues nor had data on hospitalization. Extra use of medications when infected is indicated by **. Patients with APS-1 reported between two and ten symptoms after SARS-CoV-2 infection, with a median of four ( Figures 3F and 3G). The COVID-19 symptoms were usually mild, including fever, muscle pain, fatigue, runny nose, cough, sore throat, and concentration problems, resembling a Norwegian cohort of adult vaccinated patients infected with Omicron. 25 Most patients had symptoms of COVID-19 for less than a week similarly to previous observations in healthy people of two to eight days, 25 although five subjects had persisting cough and/or fatigue for longer durations ( Figure 3H). One patient with APS-1 had two episodes with pneumonia about six months after COVID-19. Another patient complained about dyspnea six months after the infection, but later improved. One patient was hospitalized for observation while SARS-CoV-2 infected, but had no severe outcome.
During the study period, two patients died of causes unrelated to COVID-19. Eleven patients reported taking extra medications when infected; Nine with adrenal insufficiency took glucocorticoids, one with type 1 diabetes increased insulin doses, while one had hypertension and also administrated more of the active vitamin D-drug Etalpha ( Figure 1G).

DISCUSSION
In the present study, we show that COVID-vaccinated patients with APS-1 elicit antibody responses with adequate increase in SARS-CoV-2 Wuhan-specific spike IgG and neutralizing antibodies, and with sufficient levels to protect them from severe COVID-19. However, the patients with APS-1 show a senescent humoral response after vaccination similarly to elderly individuals, although probably with different underlying biological mechanisms. SARS-CoV-2 vaccination of young individuals with APS-1 has recently been shown to confer an adequate viral-specific humoral immune response. 22 Here, we confirm these findings in adult patients with APS-1. However, even though patients with APS-1 appear to have similar longitudinal decline in antibody titer as healthy controls, the lower peak response results in premature loss of functional antibodies. Hence, these patients need to be boosted with earlier and more frequent vaccine doses than healthy young individuals. As none of the patients with APS-1 included in this study reported severe adverse events after vaccination, we support previous recommendations regarding acceptable safety and tolerability of SARS-CoV-2 vaccines in immune-compromised individuals. [26][27][28][29] Notably, only two out of 10 randomly assayed patients with APS-1 and one of two healthy controls elicited low Omicron-specific neutralization antibody responses after vaccination with monovalent SARS-CoV-2 vaccines. However, two out of three patients with APS-1 sampled post Omicron infection had Omicron-neutralizing antibodies. This shows that the cross-reactive humoral Omicron response after monovalent Wuhan vaccination is modest, which has also been previously shown for healthy controls and different patient groups, explaining the breakthrough infection rates. [30][31][32][33][34][35] In the present cross-sectional design including 95% of the living Norwegian APS-1 population with 24 infected SARS-CoV-2 cases, none experienced severe illness. Only two patients complained about tiredness and dyspnea months after the infection, which might indicate delayed clearance of SARS-CoV-2 virus, but equally likely represent common post-COVID-19 symptoms. Our observations contrast with previous studies of severe COVID-19 in unvaccinated patients with APS-1, 5,6,8,15,36 although the infecting variant may have played a role. Most of our patients were infected by Omicron, which was the leading cause of COVID-19 in Norway from January 2022. 23 Alpha and Delta variants, which likely were the dominating variants in the previous studies, are less infective than Omicron, but can confer more severe disease. [37][38][39] Although mRNA vaccination offers some protection against Omicron infections (21%-44% depending on the outcome measures and time since vaccination), booster vaccinations do improve effectiveness despite variants which are able to escape vaccine-induced immunity. 40 Nevertheless, our data show that vaccination protected the patients with APS-1 against severe illness during the Omicron period. iScience Article cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells. 41 An example of the reverse situation is individuals with Down syndrome, who have initially increased expression of the receptors for IFN-Is (IFNAR1/2) encoded from chromosome 21. These individuals show higher levels of IFN-I transcripts at first, which by feedback lead to lower subsequent IFN-I responses. 42 This protects individuals with Down syndrome from SARS-CoV-2 infections but may lead to severe disease if infection does occur. We can only speculate if the IFN-autoantibodies in APS-1 (or in other IEI or elderly people) are a protective mechanism aimed at protecting internal organs from IFN-I-related tissue damage. This is underpinned by the presence of immunoreactive IFN-a in beta cells of diabetic patients and the few patients with APS-1 without IFN-a autoantibodies, which are more prone to type 1 diabetes and thyroid disease. 42,43 Such mechanisms could then be partly beneficial during COVID-19 illness but devastating for local lung tissue if the patient does not have protective immunity from vaccination or previous infection to promote SARS-CoV-2 clearance. Indeed, IFNs in general play a regulatory role in adaptive immunity, by ''Th1 skewing,'' 44 and local variations of different IFN-I subtypes may also impact viral host defense in patients with APS-1 as IFN-b and IFN-g are usually not targeted by the autoantibodies. 45 Interestingly, Hetemaki and colleagues recently showed that high levels of IFN-a4 autoantibodies in patients with APS-1 are a risk factor for severe herpesvirus infections. 46 Notably also, it has been reported that IFN-l1, being an IFN-III cytokine which is sometimes targeted by APS-1 antibodies, 47 plays a protective role in SARS-CoV-2 infections. Recently, the Omicron variant was found to induce IFN-l1, while other earlier variants did not, suggesting that IFN-l1 provides antiviral protection against Omicron in the upper respiratory tract, thus preventing spread to lower lung tissue. 48 The different functionalities and response strengths of the IFN-subtypes may hence influence the severity of SARS-CoV-2 infections. Also chemokine autoantibodies measured post-SARS-CoV-2 infection has been found to correlate with severity of COVID-19, adding to the complexity of how cytokines/chemokines interfere with host immunity toward viruses. 49 How then this plays out in patients with APS-1 with the myriad of immunoregulatory cells, cytokines, hormones, signaling molecules, and autoinflammation is yet to be determined. Notably, the IFN-I autoantibody level did not vary after the first, second, or third vaccination in our APS-1 cohort, suggesting that vaccination does not affect autoantibody responses. How this may impact upon SARS-CoV-2 virus infections is still unresolved.
In conclusion, we have shown that COVID vaccination of patients with APS-1 is safe and protects from severe outcomes of subsequent SARS-CoV-2 infections. This demonstrates that continuous presence of IFN-I autoantibodies are not major determinants for manifestations and severity of COVID-19 in these patients.

Limitations of the study
Sampling of patients who live across Norway is difficult to standardize regarding timing after vaccination, and patients were immunized with different vaccines subjected to vaccine availability. This is also the reason why T cell response studies were not possible. Our models are all based on relatively restricted numbers of observations. For example, we did not have enough samples per subject (and variation in the sample time points) to differentiate our ad-hoc model for the elderly cohort with a squared term from other models with a similar number of parameters but other decay curve shapes, so we do not report the coefficient estimates for this cohort. The relatively mild phenotypes of Norwegian patients compared to patients from Russia and USA [14,18,41,42] could reduce the generalization of our findings, although genotypically Norwegian and USA patients are similar.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
We confirm that we do not have any financial or other interest related to the submitted work that could affect or have the perception of affecting the authors objectively or could influence or have the perception of influencing the content of the article.
Plasmids with cDNA-inserts for use in radioimmunoassay and reagents for in house neutralization assay of interferons have been generated in previous studies (referred to in the methodology section) and can be available upon request and written formal MTA documents. iScience Article Data and code availability d The cytokine autoantibody and SARS-CoV-2 antibody data reported in this study cannot be deposited in a public repository because these are patient/personal data, belonging to a medical registry (for patients), the elderly and healthy controls who were health care workers. These individuals have signed informed consent forms for participation in research which we must adhere to. To request access to parts of de-identifiable data, contact Registry for organ specific autoimmune disorders (ROAS), Haukeland University hospital, Norway, e-mail: Eystein.husebye@helse-bergen.no or for elderly and healthy controls (Rebecca Cox, Rebecca.jane.cox@uib.no).

REAGENT or RESOURCE SOURCE IDENTIFIER
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Additional resources d The age-and gender-matched healthy controls and elderly who were used as comparator groups were registered in NIH clinical trials.gov (NCT04706390).

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Patients
All APS-1 patients are included in the Norwegian registry for organ-specific autoimmune disorders and have been previously described (Table S1). Diagnosis was confirmed by clinical criteria for this syndrome, AIRE mutational analysis, and/or autoantibody screening against IFN-u. 16 The study did not interfere with patient treatment, and patients used routine medication, such as glucocorticoids to restore physiological cortisol levels for those with adrenal insufficiency. One patient was receiving immune suppressive treatment (Rituximab).

Healthy controls
Age and gender-matched controls for the APS-1 patients were health care workers (control group I; n = 215, 60% females, mean age 44.5 years) 50 and healthy older adults (control group II, n = 99, 52% females, mean age 86.0 years) 52 followed pre-and post-SARS-CoV-2 vaccination ( Table 1). The control subjects were all vaccinated with two doses of BNT162b2 (Pfizer) with an interval of three weeks (21-24 days) between the first and second dose ( Table 1). None of the healthy controls received a third vaccine dose.

Ethics
All subjects provided written informed consent before inclusion in the study. Studies on APS-1 patients within the Norwegian registry for organ specific autoimmune disorders were approved by the Regional Ethical Committee (REC) of Norway (2009/2555, 2018/1417, and 285891). The age-and gender-matched healthy controls (n = 215) and elderly (n = 99) were used as comparator groups (REC: 118664 and 218629) and are registered in NIH clinical trials.gov (NCT04706390).

RIA for binding autoantibodies
Plasmids with interferon/interleukin cDNAs were used as templates in cell free protein expression experiments (in vitro transcription and translation, Promega, Madison, Wisconsin, US) to produce 35 S-labeled proteins.
For radioimmunoassay (RIA) in microtiter 96-well format, radiolabeled proteins (30000-50000 cpm per well) were mixed with serum (5 mL) in triplicates and incubated at 4 C over night. On day two, this antigen: anibody mixture was incubated with washed Protein-A-Sepharose, which will bind to the Fc-part of IgG-molecules. When applying these mixtures on filter plates (MABVN0B50, Millipore)), followed by 45 min incubation, washing and drying of the filters, levels of antbody binding can be estimated based on the radioactive signal from the bound protein to antibody, held up on the filter by the Sepharose beads. The RIAbuffer included 0.1% dithiothreitol for cytokine antibodies to prevent cross-linking and aggregation during incubation, and hence provide a more efficient immunoprecipitation. Results are expressed as binding indices ((cpm sample À cpm negative control)/(cpm positive control À cpm negative control) 3 1,000). The negative control was serum pooled from healthy blood donors, and the positive controls were from APS I patients with medium/high autoAb levels. The threshold for positivity was set as the mean of the indices of 80-150 healthy blood donors, plus three standard deviations. The methods have been described in detail previously. 12,54,55,60 The organ-specific targets for autoantibody radioimmune analysis included in Table S1 were 21-hydroxylase, 17-hydroxylase, glutamic acid decarboxylase-65, NACHT leucine-richrepeat protein 5, aromatic-L amino acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, and side-chain cleavage enzyme, and these have all been reported for the APS-I patients previously. 16 ELISA for binding cytokine autoantibodies